1. Field of the Invention
The present invention relates to a multilayer analytical element for quantitative analysis of bilirubin, and more particularly to a multilayer analytical element employable for quantitative analysis of bilirubin based on a dry system with high accuracy by a simple and quick operation.
2. Description of Prior Arts
Bilirubin, a principal component of a bile pigment in a body fluid, is produced in serum by decomposition of heme originating from hemoglobin in red blood corpuscle. Bilirubin is then absorbed by a liver, in which bilirubin is converted to a glucuronic acid-conjugated product, etc. and excreted in bile. The content of bilirubin in blood increases in response to increase of decomposition of hemoglobin as well as decrease of the liver function. Accordingly, the quantitative analysis of bilirubin is considered to be an indispensable test item in the clinical test.
As the method for quantitative analysis of bilirubin in serum, there are known a quantitative analysis method comprising photometric measurement of the yellow color inherently provided to the bilirubin, and a colorimetric analysis of red azobilirubin produced by coupling reaction of bilirubin and diazotized sulfanilate (p-sulfobenzenediazonium salt, Ehrlich reagent) based on Ehrlich reaction discovered by Van den Bergh. The latter method is generally named a diazo method.
Details of methods for quantitative analysis of bilirubin in serum are described in "Comprehensive Text of Clinical Test Technology" edited by Ishii, Vol. 6, pp. 332-350 (Igaku Shoin, 1975).
Details of the diazo method are further described below.
Bilirubin produced in serum by the decomposition of heme is named free bilirubin. This bilirubin is as such hydrophobic, but is dissolved in serum in combination with serum albumin, in the form of being adsorbed by the serum alubmin. The free bilirubin introduced into liver is combined with glucuronic acid through a covalent bond to become conjugated with glucuronic acid. Thus, a glucuronic acid-conjugated bilirubin which is improved in the water-solubility by the aid of the hydrophilic group contained in the glucuronic acid is produced. Also known is a highly water-soluble bilirubin combined to serum albumin, while no production mechanism is known on this product (J. J. Lauff, et al., Clinical Chemistry, 28(4), 629-637 (1982)).
Among these various bilirubins, the highly water-soluble conjugated bilirubin and the albumin-conjugate bilirubin both easily react with a diazonium salt, and are directly subjected to colorimetry. Accordingly, these bilirubins are named direct bilirubins.
The hydrophobic free bilirubin undergoes coupling reaction in the presence of a reaction accelerator such SO as caffeine, sodium benzoate, sodium acetate, dyphylline (C. A. Registory No. [479-18-5]), urea, a nonionic surfactant, gum arabic, an alcohol (e.g., methanol, ethanol), an acid amide, sulfoxide, etc., to produce azobilirubin. Therefore, the quantitative analysis of free bilirubin is generally performed indirectly by a stage of colorimetrically determining the total bilirubin content in a liquid sample in the presence of a reaction accelerator and a subsequent stage of subtracting the direct bilirubin content determined separately in the absence of a react.+-.on accelerator from the total bilirubin content. For this reason, the free bilirubin is otherwise named an indirect bilirubin.
Details of the diazo method for quantitative analysis of bilirubin are described in the following publications: M. Michaelsson, Scand. J. Clin. Lab. Invest., 13 (Suppl.), 1-80 (1961); H. Malloy, J. Biol. Chem., 119, 481(1939); and Z. K. Shihabi, et al., American Journal of Medical Technology, 43(10), 1004-1007(1977).
A great number of analytical processes for quantitative analysis of bilirubin based on the above-mentioned diazo method employing a multilayer analytical element have been proposed, for instance, in Japanese Patent Publication No. 53(1978)-28119, and Japanese Patent Provisional Publications Nos. 54(1979)-33094, 55(1980)- 44492, 56(1981)-10255, 56(1981)-30503, and 57(1982)-23859.
On the other hand, a number of studies on improvement of the analytical accuracy of a multilayer analytical element for quantitative analysis of bilirubin by way of utilizing a non-fibrous isotropically porous spreading layer have been already made and described in Japanese Patent Publication No. 57(1982)-25783 and Japanese Patent Provisional Publications Nos. 53(1978)-89797, 57(1982)-37262 and 57(1982)-110942. It is noted, however, that all of the multilayer analytical elements for quantitative analysis of bilirubin employing an isotropically porous spreading layer described in these publications are not based on the above-mentioned diazo method, but based on other quantitative analytical systems. Further, these known multilayer analytical elements have such drawback that non-diffusive bilirubins such as protein-conjugated bilirubin is not detectable, and accordingly the total bilirubin content is not appropriately analyzed by these analytical elements.